Interferon beta is known as a substance that has an antiviral activity and suppresses cell proliferation, and also has the functions of anti-proliferation, augmentation of the cytotoxicity of lymphocytes, nature immune regulation, macrophage activation, enhancement of cytotoxic T cell responses, and enhancement of macrophage activity, and can be used for the treatment of viral infections, autoimmune diseases, and cancer. Currently, three forms of human recombinant interferon beta have been approved by US and European regulatory authorities for the treatment of multiple sclerosis; one type of non-glycosylated serine17-interferon-beta-1b produced from a strain of Escherichia coli and two types of glycosylated interferon-beta-1a produced from mammalian cells.
Betaseron is the brand name for non-glycosylated serine17-interferon-beta-1b produced from a strain of Escherichia coli, and is given every other day at a high dose of 250 μg. Avonex (Biogen, Inc.) is the brand name for glycosylated interferon-beta-1a produced from mammalian cells, and is given once a week by intramuscular injection, and it's approval was followed by approval of Rebif (Serono, Inc.). Even though Rebif is similar to Avonex and should be given three times a week, it was approved because of differences in the efficacy and formulation.
Non-glycosylated interferon is problematic in that it is very unstable, and thus precipitation occurs due to increased sensitivity to thermal denaturation and hydrophobic aggregation. Meanwhile, glycosylated interferon has a relatively long half-life in the body, compared to non-glycosylated interferon, and the half-life is prolonged from two days (intravenous injection) to one week (intramuscular injection), depending on its route of administration. However, diseases for which interferon-beta is efficacious require chronic management, and thus a long-acting formulation capable of acting one week or longer is demanded to solve the problem of interferon-beta instability. Therefore, many efforts have been made to improve the serum stability of interferon-beta and maintain the drugs in the blood at high levels for a prolonged period of time, thereby maximizing the pharmaceutical efficacy of the drugs. These long-acting interferon-beta formulations need to increase the stability of interferon-beta and maintain the titers at sufficiently high levels without causing immune responses in patients.
To stabilize interferon-beta and prevent hydrophobic aggregation, a polymer having high solubility, such as polyethylene glycol (PEG), was conventionally used to chemically modify the surface of a peptide. By binding to specific regions of the target interferon-beta, PEG increases the molecular weight of a peptide to prevent clearance by the kidneys and aggregation and thus improves the stability to increase the in vivo half-life, without causing serious side effects. For example, WO99/55377 discloses that PEG with a size of 20 KDa is linked to the sulfhydryl group (—SH) of cysteine residue in interferon beta-1a to improve the solubility and stability at neutral pH (reduced aggregation) and to reduce immunogenicity. U.S. Pat. No. 0,962,978 B2 discloses that PEG with a size of 20, 30, or 40 KDa is linked to the N-terminus of interferon beta-1a. In addition, US 2004/0126361 discloses that PEG with a size of 10 or 30 KDa is linked to the N-terminus of interferon beta-1b. These methods increase the molecular weight of PEG to prolong the in vivo duration of a peptide drug, whereas the increased molecular weight remarkably reduces titer of the peptide drug and reactivity to interferon beta, leading to a reduction in the yield and an increase in production costs.
U.S. Pat. No. 5,908,626 describes a fusion protein of interferon beta 1a and (γ4) Fc via a peptide linker, WO200103737 describes a fusion protein of interferon beta 1a and IgG1Fc, IgG4Fc, or IgG1CH. In US600735 B2, a direct fusion of interferon beta 1a and IgG1Fc fragment is compared with a fusion via G4S linker. US20060228332 describes a fusion protein of interferon beta 1a and immunoglobulin fragment (Fc), and WO2004020405 describes a fusion protein of interferon beta and non-glycosylated transferrin (mTf). These fusion proteins are advantageous in that low pegylation yield and non-specificity can be overcome, whereas they are disadvantageous in that the serum half-life is not increased as expected, and in some cases, titer becomes low. To maximize the increase in the serum half-life, various types of peptide linkers have been used, but they may cause immune responses.
Other attempts have been made. US20040115168A1 describes glycopegylation of linking a sugar chain of interferon beta-1a with PEG, but a low pegylation yield is problematic, and Korea Patent No. 10-0541850 describes a method of adding one or more sugar chains, in which interferon beta-1a variants are used to perform partial substitution of amino acid at position 7, but the amino acid substitution of native form may generate a problem in the in vivo stability.